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Paracetamol metabolism by endothelial cells – potential mechanism underlying intravenous paracetamol-induced hypotension

ABSTRACT: We have shown previously that a metabolite of acetaminophen (APAP), N-acetyl-p-benzoquinone imine (NAPQI), is a potent vasodilator, which could underlie the hypotension observed when APAP is administered intravenously. However, it is unknown whether APAP metabolism to NAPQI is possible in the vasculature. In this study, we examine the hypothesis that APAP is metabolized by cytochrome P450 enzymes within the endothelium, which may be accelerated in critically ill patients by the presence of elevated myeloperoxidase (MPO). Exposure of human coronary artery endothelial cells (HCAECs) to APAP resulted in the formation of protein-bound APAP adducts, together with a loss of metabolic activity. Proteomic analysis of HCAECs exposed to APAP showed upregulation of CYP20A1 and cytochrome P450 reductase (POR), together with proteins involved in the pentose phosphate pathway and maintaining redox homeostasis. Furthermore, proteomic analysis of mesenteric arteries and livers from rats administered intravenous APAP showed that APAP metabolism likely took place in the vascular wall and not in the liver. Moreover, similar proteomic pathway changes were identified in HCAECs and mesenteric arteries. Intracellular thiols were depleted in HCAECs upon APAP treatment, which was partially attenuated by ketoconazole, consistent with the involvement of cytochrome P450 enzymes (CYP) in the metabolism of APAP to a thiol-reactive metabolite such as NAPQI. Evidence was also obtained for the metabolism of APAP to a thiol-reactive intermediate by MPO, in accordance with NAPQI formation. Taken together, these data provide a putative mechanism to explain the presentation of hypotension in critically ill patients following IV APAP administration.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Rattus Rattus (black Rat)

SUBMITTER: Joakim Bastrup

LAB HEAD: Thomas Jepps

PROVIDER: PXD058085 | Pride | 2025-05-20

REPOSITORIES: Pride

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Project description:Background: The vascular wall of small arteries is heavily affected by high blood pressure. However, the underlying mechanisms causing vascular changes are not fully elucidated. Using a novel data-independent acquisition mass spectrometry (DIA-MS) approach, we aimed to determine the proteomic changes in small mesenteric arteries during early-onset high blood pressure in a rat model of hypertension. Methods: Snap frozen small mesenteric and renal arteries from the spontaneous hypertension rat (SHR) model and Wistar Kyoto (WKY) control rats were collected from two time points (6- and 12-weels of age) and analyzed by a label free quantitative DIA-MS workflow. Mesenteric arteries from Wister Hannover rats were included as an additional control to clarify genetic drift caused by selective inbreeding. Results: We identified a total of 3956 consistent proteins in the mesenteric artery wall and found that 286 proteins were significantly regulated in 12-weeks old SHRs compared to WKY controls. Comparing to an in silico matrisome database, we identified 38 extracellular matrix-associated proteins that could distinguish SHRs from WKY controls. Furthermore, when comparing the significantly regulated proteins identified in mesenteric and renal arteries, we identified 18 proteins, including Serpina3l, Igg-2a, ENSRNOG00000049829, Acyp2, Enpp3, Lss, Acaa1a, Basp1, an isoform of Basp1, Flot1, Flot2, Gstt1, Nit1, Ppid, Ikbkap, Poglut3, P4ha2 and Usp15, that were changed in both vascular beds. These proteins were associated with vital cellular processes, such as dyslipidemia, protease inhibition, remodeling and generation of reactive oxygen species. Majority of the identified proteins and pathways were associated with hypertension, and mapping the underlying changes help understanding the pathological processes occurring in the arterial wall during early-onset hypertension. Conclusions: Our data provides an in-depth analysis of the proteomic architecture of the mesenteric and renal artery wall from SHRs and WKY control rats. We identified 18 novel candidate proteins that highlights critical changes in small arteries of the SHR.

Project description:Sluka2016 - Acetaminophen metabolism Liver metabolism of Acetaminophen: Acetaminophen (APAP) is metabolized in the liver in both Phase I and Phase II reactions. Phase II reactions convert APAP to APAP-glucuronide and APAP-sulfate. Phase I reactions involve Cytochrome P450 mediated (mostly Cyp450-2E1 and -1A2) conversion of APAP to N-acetyle-p-quinoneimine (NAPQI), which goes on to react with cellular nucleophiles such as glutathione (GSH). At high doses of APAP significant GSH depletion in hepatocyte occurs resulting in cell necrosis and and in extreme cases death. This model is described in the article: A Liver-Centric Multiscale Modeling Framework for Xenobiotics. Sluka JP, Fu X, Swat M, Belmonte JM, Cosmanescu A, Clendenon SG, Wambaugh JF, Glazier JA. PLoS ONE 2016; 11(9): e0162428 Abstract: We describe a multi-scale, liver-centric in silico modeling framework for acetaminophen pharmacology and metabolism. We focus on a computational model to characterize whole body uptake and clearance, liver transport and phase I and phase II metabolism. We do this by incorporating sub-models that span three scales; Physiologically Based Pharmacokinetic (PBPK) modeling of acetaminophen uptake and distribution at the whole body level, cell and blood flow modeling at the tissue/organ level and metabolism at the sub-cellular level. We have used standard modeling modalities at each of the three scales. In particular, we have used the Systems Biology Markup Language (SBML) to create both the whole-body and sub-cellular scales. Our modeling approach allows us to run the individual sub-models separately and allows us to easily exchange models at a particular scale without the need to extensively rework the sub-models at other scales. In addition, the use of SBML greatly facilitates the inclusion of biological annotations directly in the model code. The model was calibrated using human in vivo data for acetaminophen and its sulfate and glucuronate metabolites. We then carried out extensive parameter sensitivity studies including the pairwise interaction of parameters. We also simulated population variation of exposure and sensitivity to acetaminophen. Our modeling framework can be extended to the prediction of liver toxicity following acetaminophen overdose, or used as a general purpose pharmacokinetic model for xenobiotics. This model is hosted on BioModels Database and identified by: BIOMD0000000624. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

			Action	DRS
	LiverRawData.zip	Other
	MSfiles.xlsx	Xlsx
	MesentericArteryRawData.zip	Other
	UP000002494_10116.fasta	Fasta
	UP000002494_10116_additional.fasta	Fasta

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Paracetamol metabolism by endothelial cells – potential mechanism underlying intravenous paracetamol-induced hypotension

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